Cationic polysaccharides for delivery of phosphoinositides to cells for therapeutic purpose
a technology of phosphoinositide and cationic polysaccharide, which is applied in the direction of pharmaceutical non-active ingredients, organic active ingredients, pharmaceutical active ingredients, etc., can solve the problems of morbidity and mortality among diabetic patients, altered skin wound healing, and limited strategy
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example 2
PI3P Complex Characterization
[0117]The hydrodynamic radius of the Q-starch / PI3P complexes without sonication and filtration of the PI3P solution prior complexation is shown in FIG. 4A for N / P ratios of 0.5 and 1. As seen in FIG. 4A, the mean hydrodynamic radius of self-assembled Q-starch / PI3P complexes, as measured by Dynamic Light Scattering (DLS), is approximately 116 nm. The zeta potentials of these complexes are shown in FIG. 4B for N / P ratios of 0.5 and 1 and for PI3P and Q-starch alone. As seen in FIG. 4B, the zeta potential increases with increasing the N / P ratio. The hydrodynamic radius of the Q-starch / PI3P complexes prepared with sonication and filtration of the PI3P solution prior complexation is shown in FIG. 5A for N / P ratios of 1 to 5. As seen in FIG. 5A, the hydrodynamic radius of self-assembled Q-starch / PI3P complexes, as measured by Dynamic Light Scattering (DLS), inversely depends on the N / P ratio (between 254.18 nm at N / P=1 and 38.19 nm at N / P=5). The zeta potentia...
example 3
Localization of Q-Starch / PI3P Complexes
[0118]Cellular uptake of Q-starch5-DTAF / PI3P complexes (DTAF-5-(4,6-Dichlorotriazinyl) Aminofluorescein, a reactive green dye) was visualized using spinning disk confocal microscope. HEK-293 cells were seeded in 6-well culture plates on coverslips and incubated over-night at 37° C. with 5% CO2. Then the cells were supplemented with 25 μL of Q-starch5-DTAF / PI3P complexes solution (all diluted to CPI3P=0.5 μM in DMEM). Afterwards the cells were incubated for 2 hours t 37° C. with 5% CO2. The cells were fixed with 4% paraformaldehyde. Cell membrane was labeled by WGA Alexa Fluor 555.
[0119]Imaging experiments using an imaging flow cytometer (Amnis ImageStreamX) were performed to quantitatively evaluate the cellular internalization of Q-starch5-DTAF / PI3P complexes. HEK-293 cells were seeded in 6-well culture plates and incubated over-night at 37° C. with 5% CO2. Then the cells were supplemented with 25 μL of Q-starch5-DTAF / PI3P complexes solution (a...
example 4
Flux
[0121]Up-regulation of autophagy in HEK293 cells with the Q-starch / PI3P complexes was assessed by comparing the autophagy flux for the following conditions (FIG. 7): flux after 3 hours of serum starvation, flux after inhibition of the formation of endogenous PI3P by 3-MA (3-Methyladenine), flux after treatment with 3-MA and complexes at N / P ratio of 2 and flux after treatment with 3-MA and PI3P. As can be seen in FIGS. 7A-7B, inhibiting endogenous PI3P formation leads to a decrease in the autophagy flux compared to serum starvation. Treatment with Q-starch / PI3P complexes at N / P=2 succeeds to up-regulate autophagy flux even when compared to serum starvation levels. Treatment with PI3P alone did not affect the levels of autophagy flux compared to treatment with 3-MA.
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